This invention relates to improvements in the solid phase synthesis of peptides and proteins.
The classical synthesis of peptides has used the solution method in which a protected amino acid or peptide fragment has been reacted in solution with another protected amino acid or protected peptide chain. At each step of the synthesis the desired product has been separated from the reaction medium and at least partially purified before continuing to the next step. The procedure is time-consuming and tedious. However, it is capable of producing high-purity products. These procedures have been used to produce oxytocin, insulin, adrenocortocotropin and other proteins.
In 1962, Merrifield introduced the solid phase method of peptide synthesis. In this procedure, the amino acid corresponding to the carboxyl end of a peptide chain was attached to an insoluble support, normally a resin through the alpha carboxyl group and the chain was extended toward the amino end by stepwise coupling of activated amino acid derivatives. Filtration and thorough washing of the solid phase removed soluble by-products and excess reagents but retained the extended peptide chain without loss. After completion of the chain the peptide was removed from the support and purified.
The initial procedure has been modified by attaching an activated amino acid by its alpha amino group to the resin support and extending the chain towards the carboxyl end. Procedures have also been devised in which a functional group of an amino acid other than the alpha amino group or the carboxyl group has served as the link to the solid support. However, the vast majority of peptides and proteins that have been synthesized using solid phase technology have followed the original procedure in which the peptide has been extended toward the amino end. Generally the procedure is conducted by the following steps:
1. Attachment of an amino acid with a protected amino group to a resin support through its carboxyl group.
2. Removal of the protecting group from the amino group.
3. Couple the supported amino acid with a second amino acid in which the amino group is protected, by reaction between the deprotected amino group of the supported amino acid and the carboxyl group of the second amino acid.
4. Removal of the protecting group from the amino group of the second amino acid.
Steps 2-4 above are repeated as many times as is necessary to produce the desired product.
At each stage of the synthesis, the support carrying the synthesized product is recovered by filtration and thoroughly washed to remove insoluble impurities.
As those skilled in the art will be aware, there are many variations of the basic theme. For example, eleven of the twenty common amino acids bear functional groups that are usually protected during the synthesis with protecting groups which will not interfere with the overall synthesis and can be readily removed when it is desirable to do so. For example, the guanidino group of arginine has been protected by the nitro group or the 4-toluensulfonyl group. The epsilon amino group of lysine has been protected by the benzyloxylcarbonyl group. The beta carboxyl group of aspartic acid, the gamma carboxyl group of glutamic acid, the beta hydroxyl groups of serine and threonine, and the phenolic hydroxyl group of tyrosine have been protected by the benzyl group. Other protecting groups are known and have been employed to protect the extra functional groups of the named amino acids as well as other polyfunctional amino acids.
Another important variation is to increase the length of the growing chain by coupling the supported amino acid or peptide with a separately synthesized peptide fragment. Still another is to remove the peptide from the support at an intermediate stage in the synthesis, purify it and then reattach it to the support before continuing the synthesis. They have been extensively utilized to produce large numbers of peptides and proteins. Heretofore, the procedures have been as described above. The desired product has been constructed by growth originated from a single link to the selected support.
After the peptide has been assembled on a solid support, it must be removed from the support by a suitable cleavage reaction. The choice of the cleavage reagent depends primarily on the bond linking the peptide to the support. But the constituent amino acids of the peptide, the nature of their side chain protecting groups, and the structure of the final derivative desired must also be considered. Peptide removal has been effected by acidolytic or alkaline hydrolysis, aminolysis, alcoholysis and other cleavage methods. Acidolysis is by far the most widely employed procedure.
A large number of acidolytic reagents are known. These include, for example, bromine-free hydrogen bromide in trifluoracetic acid (TFA) or in acetic acid. The method of choice for most investigators is liquid hydrogen fluoride and anisole at 0.degree. C. for about one hour. A particular advantage of this reagent is that it removes all side chain protecting groups and cleaves the most widely employed anchoring bond in one step.
The most widely employed anchoring bond for the procedure in which the chain is extended toward the amino end is the ester group in which the carboxyl group of the first amino acid in the chain is attached through a benzyl group on a polystyrene support resin. The benzyl group is generated by chloromethylation of polystyrene with chloromethyl methyl ether and a Lewis acid catalyst. The first amino acid to be attached to the support is reacted as an amino-protected salt, typically the cesium salt.
A wide variety of solid supports have been studied and utilized in the solid phase synthetic methods. These include polystyrene cross-linked with divinylbenzene, phenol-formaldehyde resins, cellulose, and silicon. The support which has achieved widest acceptance is polystyrene cross-linked with about 1% to 2% divinylbenzene.
The alpha amine protecting group which has proved to be most useful in solid phase synthesis is the tertiary butyloxycarbonyl group (Boc). This group is readily substituted on the amino group and removed when necessary with minimum disruption of the peptide. However, practically all the protecting groups used in conventional peptide syntheses have been successfully employed in solid phase synthesis. These include, for example, benzyloxycarbonyl, triphenylmethyl, tertiary amyloxycarbonyl, furfuroxyloxycarbonyl and 2-nitrophenylsulfonyl.
This brief overview of the solid phase synthetic method has been provided to indicate that the procedure is well known and has been intensively studied. Principal emphasis has been on procedures in which the peptide grows towards the amino end. Procedures for extending the peptide toward the carboxyl end have also been extensively studied, but because of inherent disadvantages, such as racemization, have not been as widely employed. Those skilled in the art, however, will immediately recognize from a study of this specification that the improvements described herein are applicable to all types of solid phase peptide syntheses.
The solid phase procedures have proven to be a revolutionary advance in peptide synthesis.